Abstract We identified a chromatin-modifying factor hMOF, the human ortholog of the Drosophila MOF gene (Males absent On the First) that interacts with and regulates the ionizing radiation (IR) response function of ATM. hMOF is a member of the MYST family of histone acetyltransferases (HATs) and acetylates histone H4 at lysine 16 (H4K16ac). Histone H4K16ac is a unique histone modification that prevents higher order chromatin packing, which can impede protein access to DNA. Mammalian MOF impacts multiple points in the cellular DNA damage response (DDR) and double-strand break (DSB) repair pathways probably as a critical regulator of chromatin structure. This competing renewal brings together and expands insights gained during the previous funding period from two separate RO1?s [R01CA123232 (not being renewed), R01CA129537 (this renewal)] that focused our attention on how MOF-dependent H4K16ac levels and the phosphorylation of MOF influence DNA DSB repair and oncogenesis, as we found that acetylation of histone H4 at K16 by MOF is an epigenetic signature of cellular proliferation common to both embryogenesis and oncogenesis. In addition, our results suggest that preexisting MOF-dependent H4K16ac may influence the DDR. However, the precise effect of H4K16ac chromatin status as well as most other chromatin proteins/modifications on the DDR is largely unknown. A major impediment in the mammalian DNA repair field to answering this type of question has been the non-specificity of DNA damage inducing agents (for example: ionizing radiations), making it difficult to characterize how specific differences in chromatin environment impact DNA lesion signaling/repair. We will circumvent this problem by using site-specific repair systems to determine the role of histone H4K16ac in DSB repair. We have successfully generated a high-density genome wide map identifying H4K16ac rich or poor chromosomal sites. Utilizing this data, we will directly test the hypothesis that local H4K16ac levels on chromosomes regulate DDR. To examine sites with defined H4K16ac chromatin status, we will utilize zinc finger nucleases (ZFNs) to produce DSBs within regions we have identified as having high or low H4K16ac levels and determine the subsequent recruitment of DDR components. Furthermore, we have shown that MOF itself is phosphorylated at threonine 392 (MOF-T392) post irradiation in an ATM-dependent manner that is important for the DDR and cell survival. Therefore, to determine the role of ATM dependent MOF phosphorylation in signaling/repair protein recruitment, we will generate mutant MOF-phosphorylation (mMof- T392A) site knock-in mice and examine the DSB signaling/repair mechanism and consequences for genomic integrity and cancer development. This approach uniquely allows for the determination of how MOF-dependent local H4K16ac levels and MOF phosphorylation affect the DDR, repair pathway choice and oncogenesis.